The present invention relates to an anti-Helicobacter pylori agent comprising an oxazolin-4-one derivative useful as a therapeutic agent for gastric and duodenal ulcer etc.
As therapeutic agents for ulcer, there have been developed antacids, anticholinergic agents, antigastrin agents, gastrointestinal hormones, antipepsine agents, histamine H2 receptor antagonists, tissue repairing agents, mucosa-protecting agents, microcirculation-improving agents, proton pump inhibitors etc. The development of histamine H2 receptor antagonists and proton pump inhibitors, both possessing potent acid secretion-suppressing activity, in particular, has facilitated ulcer treatment.
However, these therapeutic agents for ulcer are unsatisfactory in terms of suppressing effect on recurrent ulcer. On the other hand, Helicobacter pylori, a gram-negative microaerophilic bacterium belonging to the genus Helicobacter, has been suggested as a potential major cause of recurrence of gastritis, duodenal ulcer, gastric ulcer etc. Although many antibacterial agents readily inhibit the proliferation of the-respective microorganisms belonging to the genus Helicobacter in vitro, their efficacy in humans and animal experiments is very low when administered singly in vivo.
Various diseases caused by Helicobacter pylori as such are treated by chemotherapies such as double chemotherapy with a bismuth preparation and an antibiotic, and triple chemotherapy with a bismuth preparation, metronidazole (U.S. Pat. No. 2,944,061) and either tetracycline (e.g., U.S. Pat. No. 2,712,517) or amoxicillin (U.S. Pat. No. 3,192,198). Metronidazole, an imidazole derivative possessing anti-Helicobacter pylori activity is used in combination with antibiotics. These bismuth preparations, antibiotics, metronidazole etc. are administered orally. Also, clinical studies have shown that eradication of this microorganism results in healing and decreased recurrence rates in ulcer.
However, these bismuth preparations, antibiotics, metronidazole etc. must be administered at high daily doses to maintain sufficient concentrations to inhibit Helicobacter pylori proliferation at the sites of their proliferation, resulting in many problems, including adverse effects such as vomiting and diarrhea.
There have been developed various compounds possessing anti-Helicobacter pylori activity. For example, Japanese Patent Unexamined Publication No. 117268/1993 discloses a pyridine derivative possessing anti-Helicobacter pylori activity, and European Patent EPO 535528A1 discloses an imidazole derivative possessing anti-Helicobacter pylori activity.
After extensive investigation in view of the above problems, the present inventors found that a particular oxazolin-4-one derivative exhibits very specific and excellent antibacterial activity against the bacteria of the genus Helicobacter, represented by Helicobacter pylori. The inventors conducted further investigation based on this finding, and developed the present invention.
Accordingly, the present invention relates to
(1) an anti-Helicobacter pylori composition comprising a compound of the formula: 
xe2x80x83wherein A represents an aromatic group which may be substituted, R1 and R2 independently represent a hydrogen atom or a hydrocarbon group which may be substituted, R3 and R4 independently represent a hydrogen atom, a hydrocarbon group which may be substituted, an acyl group, a carbamoyl group which may be substituted, or a carboxyl group which may be esterified, or a salt thereof, and a pharmacologically acceptable diluent, excipient or carrier,
(2) the anti-Helicobacter pylori composition according to the description in (1) above, wherein A is an aromatic heterocyclic group which may be substituted,
(3) the anti-Helicobacter pylori composition according to the description in (1) above, wherein A is a group represented by the formula: 
xe2x80x83wherein ring B is a 6-membered aromatic ring which may be substituted, x represents CH or N, Y represents O, S or xe2x80x94Nxe2x80x94R5 (R5 represents a hydrogen atom or a hydrocarbon group which may be substituted),
(4) the anti-Helicobacter pylori composition according to the description in (1) above, wherein A is a group represented by the formula: 
xe2x80x83wherein ring B is a 6-membered aromatic ring which may be substituted, R5 represents a hydrogen atom or a hydrocarbon group which may be substituted,
(5) the anti-Helicobacter pylori composition according to the description in (1) above, wherein A represented indolyl which may be substituted by 1 to 3 substituents selected from the group consisting of hydroxyl, halogen, nitro, cyano, lower alkyl which may be substituted by 1 to 5 halogens and lower alkoxy which may be substituted by 1 to 5 halogens, R1 and R2 independently represent hydrogen or lower alkyl which may be substituted by 1 to 5 halogens, R3 and R4 independently represent hydrogen or lower alkyl,
(6) the anti-Helicobacter pylori composition according to the description in (5) above, wherein A is indolyl, R1 and R3 are hydrogen, and R2 and R4 are C1-7 alkyl,
(7) the anti-Helicobacter pylori composition according to the description in (6) above, wherein A is 3-indolyl, R2 and R4 are methyl,
(8) the anti-Helicobacter pylori composition according to the description in (1) above, wherein the compound is indolmycin,
(9) the anti-Helicobacter pylori composition according to the description in (1) above, as an agent for prevention or treatment of a disease associated with Helicobacter pylori infection,
(10) the anti-Helicobacter pylori composition according to the description in (9) above, wherein the disease associated with Helicobacter pylori infection is gastric or duodenal ulcer, gastritis or gastric cancer,
(11) the anti-Helicobacter pylori composition according to the description in (1) above, which is used in combination with an antibacterial agent,
(12) the anti-Helicobacter pylori composition according to the description in (1) above, which is used in combination with antiulcer agent,
(13) the anti-Helicobacter pylori composition according to the description in (1) above, which is used in combination with antibacterial agent and antiulcer agent,
(14) use of compound of the formula: 
xe2x80x83wherein A represents an aromatic group which may be substituted, R1 and R2 independently represent a hydrogen atom or a hydrocarbon group which may be substituted, R3 and R4 independently represent a hydrogen atom, a hydrocarbon group which may be substituted, an acyl group a carbamoyl group which may be substituted, or a carboxyl group which may be esterified, or a salt thereof for the preparation of an anti-Helicobacter pylori agent,
(15) a method for prevention or treatment of a disease associated with Helicobacter pylori infection in a mammal which comprises administering to a subject in need an effective amount of a compound of the formula (I): 
xe2x80x83wherein A represents an aromatic group which may be substituted, R1 and R2 independently represent a hydrogen atom or a hydrocarbon group which may be substituted, R3 and R4 independently represent a hydrogen atom, a hydrocarbon group which may be substituted, an acyl group, a carbamoyl group which may be substituted, or a carboxyl group which may be esterified, or a salt thereof,
(16) a method for producing an anti-Helicobacter pylori composition comprising mixing a compound of the formula (I): 
xe2x80x83wherein A represents an aromatic group which may be substituted, R1 and R2 independently represent a hydrogen atom or a hydrocarbon group which may be substituted, R3 and R4 independently represent a hydrogen atom, a hydrocarbon group which may be substituted, an acyl group, a carbamoyl group which may be substituted, or a carboxyl group which may be esterified, or a salt thereof with a pharmacologically acceptable diluent, excipient or/and carrier,
xe2x80x83(17) a compound of the formula: 
xe2x80x83wherein A represents an aromatic group which may be substituted, R1 and R2 independently represent a hydrogen atom or a hydrocarbon group which may be substituted, R3xe2x80x2 and R4xe2x80x2 independently represent a hydrogen atom or a hydrocarbon group which may be substituted, or a salt thereof, provided that (1) when A is 3-indolyl, R1 and R3xe2x80x2 are hydrogen and R2 is methyl, R4xe2x80x2 is neither C3-6 cycloalkyl nor mono-substituted C1-4 alkyl wherein said substituent is selected from halogen, hydroxyl, lower alkoxy, lower thioalkyl, aryl, or an unsaturated 2-4 carbon atoms side-chain and (2) when A is 3-indolyl, R1 and R3xe2x80x2 are hydrogen and R2 is C1-3 alkyl, R4xe2x80x2 is not selected from hydrogen, phenyl, anisyl, toluidyl and C1-4 alkyl,
(18) a compound of the formula: 
xe2x80x83wherein A represents an aromatic group which may be substituted, R1 and R2 independently represent a hydrogen atom or a hydrocarbon group which may be substituted, R3xe2x80x2 is a hydrogen atom or a hydrocarbon group which may be substituted, R4xe2x80x3 is an acyl group or a carbamoyl group which may be substituted or a salt thereof, provided that when A is 3-indolyl, R1 is hydrogen and R2 and R3xe2x80x2 are methyl, R4xe2x80x3 is neither a C2-5 alkanoyl or an mono-substituted C2-5 alkanoyl wherein said substituent is selected from amino, halogen, phenyl, p-hydroxyphenyl, or lower alkoxy, nor a carbamoyl group substituted by C1-4 alkyl, C3-6 cycloalkyl or phenyl,
(19) a compound of the formula: 
xe2x80x83wherein A represents an aromatic group which may be substituted, R1 and R2 independently represent a hydrogen atom or a hydrocarbon group which may be substituted, R3xe2x80x2 is a hydrogen atom or a hydrocarbon group which may be substituted, R4xe2x80x2xe2x80x3 is a carboxyl group which may be esterified, or a salt thereof,
(20) the compound according to the description in (19) above, wherein A is indolyl, R1 and R2 independently represent a hydrogen atom or methyl, R3xe2x80x2 is methyl and R4xe2x80x2xe2x80x3 is a carboxyl group which is esterified,
(21) a method of producing indolmycin by culturing the Streptomyces sp. HC-21 strain in a medium to produce and accumulate indolmycin in the culture broth, and harvesting the indolmycin, and
(22) the Streptomyces sp. HC-21 strain which assimilates L-rhamnose and whose spores have a spiny surface.
The xe2x80x9caromatic ring group which may be substitutedxe2x80x9d represented by A in formula (I) is exemplified by monocyclic or condensed polycyclic aromatic hydrocarbon groups or aromatic heterocyclic groups. Such aromatic hydrocarbon groups include, for example, phenyl, naphthyl, anthryl, phenanthryl and acenaphthylenyl, with preference given to phenyl, 1-naphthyl, 2-naphthyl etc.
The aromatic heterocyclic groups include, for example, aromatic monocyclic heterocyclic groups such as furyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, furazanyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl and triazinyl; and aromatic condensed heterocyclic groups such as benzofuranyl, isobenzofuranyl, benzo[b]thienyl, indolyl, isoindolyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, 1,2-benzisoxazolyl, benzothiazolyl, 1,2-benzisothiazolyl, 1H-benzotriazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, naphthyridinyl, purinyl, pteridinyl, carbazolyl, xcex1-carbolinyl, xcex2-carbolinyl, xcex3-carbolinyl, acridinyl, phenoxazinyl, phenothiazinyl, phenazinyl, phenoxathiinyl, thianthrenyl, phenanthridinyl, phenanthrolinyl, indolizinyl, pyrrolo[1,2-b]pyridazinyl, pyrazolo[1,5-a]pyridyl, imidazo[1,2-a]pyridyl, imidazo[1,5-a]pyridyl, imidazo[1,2-b]pyridazinyl, imidazo[1,2-a]pyrimidinyl, 1,2,4-triazolo[4,3-a]pyridyl and 1,2,4-triazolo[4,3-b]pyridazinyl. Among aromatic condensed heterocyclic group, indolyl is preferable and 3-indolyl is more preferable.
Substituents for the xe2x80x9caromatic ring group or aromatic heterocyclic group which may be substitutedxe2x80x9d represented by A in formula (I) include, for example, hydroxyl group, halogens (e.g., fluorine, chlorine, bromine, iodine), nitro, cyano, lower alkyls that may be substituted by 1 to 5 halogens (e.g., fluorine, chlorine, bromine, iodine), lower alkoxys that may be substituted by 1 to 5 halogens (e.g., fluorine, chlorine, bromine, iodine) benzyloxy and C1-4 alkoxy carbonyl (e.g. methoxy carbonyl, ethoxy carbony, propoxy carbonyl, butoxy carbonyl). Such lower alkyls include, for example, alkyl groups having 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl, with preference given to methyl and ethyl. Such lower alkoxys include alkoxy groups having 1 to 4 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy, with preference given to methoxy and ethoxy. It is preferable that 1 to 3 (preferably 1 to 2) of these substituents, whether identical or not, be present. Substituents for the xe2x80x9caromatic ring group or aromatic heterocyclic group which may be substitutedxe2x80x9d represented by A also include alkylene dioxo such as methylene dioxo and ethylene dioxo.
The xe2x80x9chydrocarbon group which may be substitutedxe2x80x9d represented by R1 or R2 in formula (I) include aliphatic chain hydrocarbon groups, alicyclic hydrocarbon groups and aryl groups, with preference given to aliphatic chain hydrocarbon groups.
Such aliphatic chain hydrocarbon groups include linear or branched aliphatic hydrocarbon groups such as alkyl groups, alkenyl groups and alkynyl groups. Particularly preferred are lower alkyl groups, lower alkenyl groups, lower alkynyl groups etc. Such lower alkyls include, for example, C1-7 alkyls such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 1-methylpropyl, n-hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 3,3-dimethylpropyl, 2-ethylbutyl and n-heptyl. Preferred are C1-3 alkyls such as methyl, ethyl and propyl, with greater preference given to C1-2 alkyls such as methyl and ethyl. Such lower alkenyl groups include, for example, C2-6 alkenyl groups such as vinyl, allyl, isopropenyl, 2-methylallyl, 1-propenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-ethyl-1-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl and 5-hexenyll with preference given to C2-5 alkenyls such as vinyl, allyl, isopropenyl, 2-methylallyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl and 3-methyl-2-butenyl. Such lower alkynyl groups include, for example, C2-6 alkynyls such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl, with preference given to C2-4 alkynyls such as ethynyl, 1-propynyl and 2-propynyl.
Such alicyclic hydrocarbon groups include saturated or unsaturated alicyclic hydrocarbon groups such as cycloalkyl groups, cycloalkenyl groups and cycloalkadienyl groups. Such cycloalkyl groups are preferably cycloalkyl groups having 3 to 9 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and cyclononyl, with greater preference given to C3-6 cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Such cycloalkenyl groups include, for example, C3-6 cycloalkenyls such as 2-cyclopenten-1-yl, 3-cyclopenten-1-yl, 2-cyclohexen-1-yl, 3-cyclohexen-1-yl, 1-cyclobuten-1-yl and 1-cyclopenten-1-yl. Such cycloalkadienyl groups include, for example, C4-6 cycloalkadienyls such as 2,4-cyclopentadien-1-yl, 2,4-cyclohexadien-1-yl and 2,5-cyclohexadien-1-yl.
The aryl groups in the hydrocarbon groups include monocyclic or condensed polycyclic aromatic hydrocarbon groups such as phenyl, naphthyl, anthryl, phenanthryl and acenaphthylenyl, with preference given to phenyl, 1-naphthyl, 2-naphthyl etc.
Substituents for the xe2x80x9chydrocarbon group which may be substitutedxe2x80x9d represented by R1 or R2 in formula (I) include aryl groups which may be substituted, cycloalkyl or cycloalkenyl groups which may be substituted, heterocyclic groups that may be substituted, amino group that may be substituted, hydroxyl group which may be substituted, thiol group which may be substituted, and halogens (e.g., fluorine, chlorine, bromine, iodine). One to five (preferably 1 to 3) of these optionally chosen substituents may be present. Such aryl groups which may be substituted include phenyl, naphthyl, anthryl, phenanthryl and acenaphthylenyl, with preference given to phenyl, 1-naphthyl and 2-naphthyl. Substituents for such aryl groups which may be substituted include alkoxy groups having 1 to 3 carbon atoms (e.g., methoxy, ethoxy, propoxy), halogen atoms (e.g., fluorine, chlorine, bromine, iodine) and alkyl groups having 1 to 3 carbon atoms (e.g., methyl, ethyl, propyl); 1 to 2 of these optionally chosen substituents may be present. Such cycloalkyl groups which may be substituted include C3-7 cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. The kinds and number of substituents for such cycloalkyl groups which may be substituted are the same as those for the substituents the above-described aryl group that may be substituted. Such cycloalkenyl groups which may be substituted include C3-6 cycloalkenyl groups such as cyclopropanyl, cyclobutenyl, cyclopentenyl and cyclohexenyl. The kinds and number of substituents for such cycloalkenyl groups which may be substituted are the same as those for the substituents for the above-described aryl group that may be substituted. Such heterocyclic groups which may be substituted for include aromatic heterocyclic groups having at least 1 hetero atom selected from oxygen, sulfur or nitrogen as a ring-constituting atom (ring atom), and saturated or unsaturated non-aromatic heterocyclic groups (aliphatic heterocyclic groups), with preference given to aromatic heterocyclic groups. Such aromatic heterocyclic groups include aromatic monocyclic heterocyclic groups (e.g., furyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, furazanyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl) and aromatic condensed heterocyclic groups (e.g., benzofuranyl, isobenzofuranyl, benzo[b]thienyl, indolyl, isoindolyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, 1,2-benzisoxazolyl, benzothiazolyl, 1,2-benzisothiazolyl, 1H-benzotriazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, naphthyridinyl, purinyl, pteridinyl, carbazolyl, xcex1-carbolinyl, xcex2-carbolinyl, xcex3-carbolinyl, acridinyl, phenoxazinyl, phenothiazinyl, phenazinyl, phenoxathiinyl, thianthrenyl, phenanthridinyl, phenanthrolinyl, indolizinyl, pyrrolo[1,2-b]pyridazinyl, pyrazolo[1,5-a]pyridyl, imidazo[1,2-a]pyridyl, imidazo[1,5-a]pyridyl, imidazo[1,2-b]pyridazinyl, imidazo[1,2-a]pyrimidinyl, 1,2,4-triazolo[4,3-a]pyridyl, 1,2,4-triazolo[4,3-b]pyridazinyl), with preference given to furyl, thienyl, indolyl, isoindolyl, pyrazinyl, pyridyl, pyrimidinyl etc. Such non-aromatic heterocyclic groups include, for example, oxylanyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, tetrahydrofuryl, thiolanyl, piperidyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl and piperazinyl. Substituents for such heterocyclic groups which may be substituted for include alkyl groups having 1 to 3 carbon atoms (e.g., methyl, ethyl, propyl). Substituents for such amino group which may be substituted for, hydroxyl group that may be substituted for, and thiol group that may be substituted for, include, for example, lower (C1-3) alkyl groups (e.g., methyl, ethyl, propyl). When the xe2x80x9chydrocarbon group which may be a substitutedxe2x80x9d represented by R1 or R2 is an alicyclic hydrocarbon group or an aryl group, the substituent may also be an alkyl group having 1 to 3 carbon atoms (e.g., methyl, ethyl, propyl).
With respect to the formula (I), the preferable combination of R1 and R2 is that R1 is hydrogen and R2 is C1-3 alkyl which may be substituted by 1 to 5 halogens.
The hydrocarbon group and the substituent in the xe2x80x9chydrocarbon group which may be substitutedxe2x80x9d represented by R3 or R4 in formula (I) and represented by R3xe2x80x2 or R4xe2x80x2 in formula (Ixe2x80x2) are exemplified by the same hydrocarbon groups and substituents mentioned to exemplify the hydrocarbon group and substituent for R1 and R2 above, respectively.
With respect to the formula (I), the preferable combination of R3 and R4 is that R3 is hydrogen and R4 is C1-3 alkyl.
The acyl group represented by R3 or R4 in formula (I) is exemplified by aliphatic acyl groups such as alkanoyl groups, alkenoyl groups, cycloalkanecarbonyl groups and alkanesulfonyl groups; aromatic acyl groups such as aroyl groups, arylalkanoyl groups, arylalkenoyl groups and arenesulfonyl groups; heterocyclic aromatic acyl groups such as aromatic heterocyclic carbonyl groups and aromatic heterocyclic alkanoyl groups; and non-aromatic heterocyclic arbonyl groups (aliphatic heterocyclic carbonyl groups).
xe2x80x9cAlkanoyl groupsxe2x80x9d mean alkylcarbonyl groups, preferable examples thereof including lower alkanoyl groups having 1 to 8 carbon atoms, such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl and hexanoyl.
xe2x80x9cAlkenoyl groupsxe2x80x9d mean alkenylcarbonyl groups, preferable examples thereof including C3-6 alkenoyl groups such as acryloyl, methacryloyl, crotonoyl and isocrotonoyl.
xe2x80x9cCycloalkanecarbonyl groupsxe2x80x9d mean cycloalkylcarbonyl groups, preferable examples thereof including those having 4 to 7 carbon atoms, such as cyclopropanecarbonyl groups, cyclobutanecarbonyl groups, cyclopentanecarbonyl groups and cyclohexanecarbonyl groups.
xe2x80x9cAlkanesulfonyl groupsxe2x80x9d mean alkylsulfonyl groups, preferable examples thereof including those having 1 to 4 carbon atoms, Such as mesyl, ethanesulfonyl and propanesulfonyl.
xe2x80x9cAroyl groupsxe2x80x9d mean arylcarbonyl groups, preferable examples thereof including those having 7 to 11 carbon atoms, such as benzoyl, p-toluoyl, 1-naphthoyl and 2-naphthoyl.
xe2x80x9cArylalkanoyl groupsxe2x80x9d mean alkylcarbonyl groups substituted for by an aryl group, preferable examples thereof including C6-8 aryl-C2-5 alkanoyl groups such as phenylacetyl, phenylpropionyl, hydroatropoyl and phenylbutyryl.
xe2x80x9cArylalkenoyl groupsxe2x80x9d mean alkenylcarbonyl groups substituted for by an aryl group, preferable examples thereof including C6-8 aryl-C3-5 alkenoyl groups such as cinnamoyl and atropoyl.
xe2x80x9cArenesulfonyl groupsxe2x80x9d mean arylsulfonyl groups, preferable examples thereof including those having 6 to 8 carbon atoms, such as benzenesulfonyl and p-toluenesulfonyl.
Preferable examples of xe2x80x9caromatic heterocyclic carbonyl groupsxe2x80x9d include furoyl, thenoyl, nicotinoyl, isonicotinoyl, pyrrolecarbonyl, oxazolecarbonyl, thiazolecarbonyl, imidazolecarbonyl and pyrazolecarbonyl.
xe2x80x9cAromatic heterocyclic alkanoyl groupsxe2x80x9d mean alkylcarbonyl groups substituted by an aromatic heterocyclic group, preferable examples thereof including aromatic heterocyclic ring-C2-5 alkanoyl groups such as thienylacetyl, thienylpropanoyl, furylacetyl, thiazolylacetyl, 1,2,4-thiadiazolylacetyl and pyridylacetyl.
Preferable examples of xe2x80x9cnon-aromatic heterocyclic carbonyl groupsxe2x80x9d include aliphatic heterocyclic carbonyls such as azetidinylcarbonyl, pyrrolidinylcarbonyl and piperidinylcarbonyl.
The carbamoyl group which may be substituted represented by R3 or R4 in formula (I) and represented by R4xe2x80x2 in formula (Ixe2x80x2), is exemplified by xe2x80x9cN-monosubstitutional carbamoyl groupsxe2x80x9d and xe2x80x9cN,N-disubstitutional carbamoyl groups,xe2x80x9d as well as non-substitutional carbamoyl. An xe2x80x9cN-monosubstitutional carbamoyl groupxe2x80x9d means a carbamoyl group having one substituent on nitrogen. Examples of said substituent include C1-6 alkyls (e.g., methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, hexyl), C3-6 cycloalkyl groups (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl), aryl groups (e.g., phenyl, 1-naphthyl, 2-naphthyl), aralkyl groups (e.g., benzyl, phenethyl) and heterocyclic groups (e.g., the xe2x80x9cheterocyclic groupsxe2x80x9d mentioned to exemplify the xe2x80x9csubstituentxe2x80x9d for the xe2x80x9chydrocarbon residue which may be substitutedxe2x80x9d represented by R1 or R2 above). Such aryl groups, aralkyl groups and heterocyclic groups may be substituted. Said substituent is exemplified by hydroxyl group, amino group which may substituted by 1 or 2 lower alkyls (e.g., those having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl and butyl) or acyl groups (e.g., formyl, acetyl, propionyl, benzoyl), halogens (e.g., fluorine, chlorine, bromine, iodine), nitro, cyano, lower alkyls which may be substituted by 1 to 5 halogens (e.g., fluorine, chlorine, bromine, iodine) and lower alkoxys which may be substituted by 1 to 5 halogens (e.g., fluorine, chlorine, bromine, iodine). Such lower alkyls include, for example, alkyl groups having 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl, with preference given to methyl and ethyl. Such lower alkoxys include alkoxy groups having 1 to 4 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy, with preference given to methoxy and ethoxy. It is preferable that 1 to 3 (preferably 1 to 2) of these substituents, whether identical or not, be present.
An xe2x80x9cN,N-disubstitutional carbamoyl groupxe2x80x9d means a carbamoyl group having two substituents on a nitrogen atom. Examples of one of said substituents include the same substituents as those mentioned to exemplify the substituent for the xe2x80x9cN-monosubstitutional carbamoyl groupxe2x80x9d above; examples of the other substituent include C1-6 alkyl groups (e.g., methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, hexyl), C3-6 cycloalkyl groups (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl) and C6-10 aralkyl groups (e.g., benzyl, phenethyl). The two substituents may form a cyclic amino group in cooperation with the nitrogen atom. In this case, examples of cyclic aminocarbamoyl groups include 1-azetidinylcarbonyl, 1-pyrrolidinylcarbonyl, piperidinocarbonyl, morpholinocarbonyl, 1-piperazinylcarbonyl, and 1-piperazinylcarbonyl having a lower alkyl group such as C1-6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, hexyl), an aralkyl group such as benzyl and phenethyll an aryl group such as phenyl, 1-naphthyl and 2-naphthyl, or the like, at the 4-position.
The xe2x80x9ccarboxyl group which may be esterifiedxe2x80x9d represented by R3 or R4 in formula (I) and represented by R4xe2x80x2xe2x80x3 in formula (Ixe2x80x2xe2x80x3), is exemplified by xe2x80x9clower alkoxycarbonyl groups,xe2x80x9d xe2x80x9caryloxycarbonyl groupsxe2x80x9d and xe2x80x9caralkyloxycarbonyl groups,xe2x80x9d as well as free carboxyl group.
Preferable examples of xe2x80x9clower alkoxycarbonyl groupsxe2x80x9d include those having 2 to 8 carbon atoms, such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, sec-butoxycarbonyl, tert-butoxycarbonyl, pentyloxycarbonyl, isopentyloxycarbonyl, neopentyloxycarbonyl and tert-pentyloxycarbonyl, with preference given to those having 2 to 4 carbon atoms, such as methoxycarbonyl, ethoxycarbonyl and propoxycarbonyl.
Preferable examples of xe2x80x9caryloxycarbonyl groupsxe2x80x9d include those having 7 to 12 carbon atoms, such as phenoxycarbonyl, 1-naphthoxycarbonyl and 2-naphthoxycarbonyl. Preferable examples of xe2x80x9caralkyloxycarbonyl groupsxe2x80x9d include those having 8 to 10 carbon atoms, such as benzyloxycarbonyl and phenethyloxycarbonyl. These aryloxycarbonyl groups and aralkyloxycarbonyl groups may be substituted; useful substituents are identical to those mentioned to exemplify the substituent for aryl groups and aralkyl groups in the case of N-monosubstitutional carbamoyl groups.
The xe2x80x9c6-membered aromatic ring which may be substitutedxe2x80x9d represented by ring B in formula (II) is exemplified by benzene ring which may be substituted, and 6-membered aromatic heterocyclic ring that may be substituted. When ring B represents a benzene ring which may be substituted, formula (II) represents a group represented by the formula: 
wherein ring C may be substituted; X and Y have the same definitions as those shown above. When ring B represents a 6-membered aromatic heterocyclic ring which may be substituted, the groups represented by formula (II) include, for example, those represented by the following formulas: 
In these formulas, ring D may be substituted; X and Y have the same definitions as those shown above.
With respect to the above formulas, the substituents for rings C and D are identical to those mentioned to exemplify the xe2x80x9csubstituentxe2x80x9d for the xe2x80x9caromatic ring group which may be substitutedxe2x80x9d, represented by A. These substituents may bound to any carbon atom of rings C and D.
The xe2x80x9chydrocarbon group which may be substitutedxe2x80x9d represented by R5 in formula (II-1) is exemplified by the same hydrocarbon groups as those mentioned to exemplify the hydrocarbon group represented by R1 or R2, which may be substituted.
With respect to the formula (I), the preferable combination of A, R1, R2, R3 and R4 is that A is indolyl, R1 and R3 are hydrogen, and R2 and R4 are C1-3 alkyl. The specific examples of the above-described oxazolin-4-one derivative include indolmycin.
Salts of the compound represented by formula (I), (Ixe2x80x2), (Ixe2x80x3) or (Ixe2x80x2xe2x80x3) include pharmacologically acceptable acid addition salts; acids that form an acid addition salt include acetic acid, lactic acid, succinic acid, maleic acid, tartaric acid, citric acid, gluconic acid, ascorbic acid, benzoic acid, methanesulfonic acid, p-toluenesulfonic acid, cinnamic acid, fumaric acid, phosphonic acid, hydrochloric acid, hydrogen bromide, hydrogen iodide, sulfamic acid and sulfonic acid.
Examples of the compound represented by the formula (I) are given below.
The above compounds may be racemates or optical isomers.
Compound (I) or a salt thereof, used for the present invention, is effective as an antibacterial agent in the prevention or treatment of xe2x80x9cduodenal ulcer, gastric ulcer, gastritis (including chronic gastritis), gastric cancer etc.xe2x80x9d caused by Helicobacter pylori infection as described above, because it possesses antibacterial activity, especially potent antibacterial activity against the bacteria of the genus Helicobacter, represented by Helicobacter pylori. 
The preparation of the present invention, containing compound (I) or a pharmacologically acceptable salt thereof, can be orally or non-orally administered as an antibacterial or antiulcer agent to mammals (e.g., humans, dogs, cats, monkeys, rats, mice, horses, bovines), oral administration being normally preferred.
Examples of dosage forms for oral administration include tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions and suspensions. Examples of dosage forms for non-oral administration include injectable preparations, infusions, drip infusions and suppositories.
The content of compound (I) or a salt thereof in the preparation of the present invention is normally 2 to 85% by weight, preferably 5 to 70% by weight.
For preparing compound (I) or a salt thereof in the above-mentioned dosage forms, known production methods in common use in relevant fields are applicable. In producing the above-mentioned dosage forms, excipients, binders, disintegrants, lubricants, sweetening agents, surfactants, suspending agents, emulsifiers etc. in common use in the field of pharmaceutical making may be added in appropriate amounts as necessary.
When compound (I) or a salt thereof is prepared as tablets, for example, excipients, binders, disintegrants, lubricants etc. may be contained; when compound (I) or a salt thereof is prepared as pills or granules, excipients, binders, disintegrants etc. may be contained. When, compound (I) or a salt thereof is prepared as powders or capsules, excipients etc. may be contained; when compound (I) or a salt thereof is prepared as syrups, sweetening agents etc. may be contained; when compound (I) or a salt thereof is prepared as emulsions or suspensions, suspending agents, surfactants, emulsifiers etc. may be contained. Examples of excipients include lactose, saccharose, glucose, starch, sucrose, microcrystalline cellulose, powdered glycyrrhiza, mannitol, sodium hydrogen carbonate, calcium phosphate and calcium sulfate. Examples of binders include 5-10% by weight starch glue solutions, 10-20% by weight gum arabic solutions or gelatin solutions, 1-5% by weight tragacanth solutions, carboxymethyl cellulose solutions, sodium alginate solutions and glycerol. Examples of disintegrants include starch and calcium carbonate. Examples of lubricants include magnesium stearate, stearic acid, calcium stearate and purified talc. Examples of sweetening agents include glucose, fructose, invert sugar, sorbitol, xylitol, glycerol and simple syrups. Examples of surfactants include sodium lauryl sulfate, polysorbate 80, sorbitan monofatty acid ester and stearic acid polyoxyl 40. Example of suspending agents include gum arabic, sodium alginate, carboxymethyl cellulose sodium, methyl cellulose and bentonite. Examples of emulsifiers include gum arabic, tragacanth, gelatin and polysorbate 80.
For preparing compound (I) or a salt thereof in the above-mentioned dosage forms, coloring agents, preservatives, flavoring agents, correctives, stabilizers, thickening agents etc. in common use in the field of pharmaceutical making may be added in appropriate amounts as desired. The preparation of the present invention, which contains a compound represented by general formula (I) or a pharmaceutically acceptable salt thereof, is stable and of low toxicity, and can be safely used. Varying depending on patient condition and body weight, kind of compound, route of administration etc., the daily dose of the preparation of the present invention is normally 1 to 500 mg, preferably about 10 to 200 mg, based on active ingredient content (compound (I) or a salt thereof), per adult (weighing about 60 kg) for oral administration in patients with gastric ulcer caused by Helicobacter pylori infection.
Within the above-described dose range, no toxicity is seen.
Also, in the preparation of the present invention, compound (I) or a salt thereof can be used in combination with other antibacterial agents and antiulcer agents.
Other antibacterial agents that can be used in combination with compound (I) or a salt thereof include, for example, nitroimidazole antibiotics (e.g., tinidazole and metronidazole), tetracyclines (e.g., tetracycline, doxycycline and minocycline), penicillins (e.g., amoxicillin, ampicillin and mezlocillin), cephalosporins (e.g., cefaclor, cefadroxil, cefazolin, cefuroxime, cefuroxime axetil, cephalexin, cefpodoxime proxetil, ceftazidime and ceftriaxone), carbapenems (e.g., imipenem and meropenem), aminoglycosides (e.g., paromomycin), macrolide antibiotics (e.g., erythromycin, clarithromycin and azithromycin), lincosamide antibiotics (e.g., clindamycin), rifamycins (e.g., rifampicin) and quinolone antibiotics (e.g., ciprofloxacin, ofloxacin) nitrofurantoin. Antiulcer agents that can be used in combination with compound (I) or a salt thereof include, for example, proton pump inhibitors (e.g., omeprazole, lansoprazole, pantoprazole, rabeprazole) Histamine H2 antagonists (e.g., ranitidine, cimetidine and famotidine), and mucosa-protecting antiulcer agents (e.g., sofalcone, plaunotol, teprenone, sucralfate).
The above-described other antibacterial agents and antiulcer agents may be used in combination of two or more kinds. In this case, the dose of antibacterial agent is normally 1 to 500 mg, preferably 5 to 200 mg, per adult per day in oral administration; the dose of antiulcer agent is normally 0.5 to 1,000 mg, preferably 1 to 500 mg, per adult per day in oral administration.
The compound of formula (I) or a salt thereof can, for example, be produced by methods A through E below. 
In the above formulas, Z represents a halogen atom or xe2x80x94Oxe2x80x94SO2R6 (R6 represents a lower alkyl group or a substituted phenyl group); the other symbols have the same definitions as those shown above.
The halogen atom represented by Z in formula (III) is exemplified by fluorine, chlorine, bromine and iodine. The lower alkyl group represented by R6 is exemplified by alkyl groups having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl and 2-ethylbutyl, with preference given to those having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl and isobutyl.
Useful substituents for the substituted phenyl group represented by R6 include, for example, lower alkyl groups (same as those mentioned to exemplify the lower alkyl group represented by R6 above), lower alkoxy groups (e.g., those having 1 to 4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy and butoxy), halogen atoms (e.g., fluorine, chlorine, bromine, iodine), nitro groups, cyano groups and carboxyl groups.
This method is conducted by reacting compound (III) or a salt thereof with compound (IV) in the presence of a base. The salt of compound (III) is exemplified by the acid addition salts mentioned to exemplify acids that form an acid addition salt with compound (I). This reaction is normally carried out in a solvent; a solvent that does not interfere with the reaction is chosen as appropriate. Such solvents include, for example, alcohols such as methanol, ethanol, propanol, isopropanol, butanol and tert-butanol; ethers such as dioxane, tetrahydrofuran, diethyl ether, tert-butyl methyl ether, diisopropyl ether and ethylene glycol-dimethyl ether; esters such as ethyl formate, ethyl acetate and n-butyl acetate; halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, trichlene and 1,2-dichloroethane; hydrocarbons such as n-hexane, benzene and toluene; amides such as formamide, N,N-dimethylformamide and N,N-dimethylacetamide; ketones such as acetone, methyl ethyl ketone and methyl isobutyl ketone; nitrites such as acetonitrile and propionitrile; dimethyl sulfoxide, sulfolane, hexamethylphosphoramide and water; these solvents are used as simple or mixed solvents.
Useful bases include, for example, C1-6 alkyl or aryl lithiums such as methyl lithium, ethyl lithium, n-butyl lithium, sec-butyl lithium, tert-butyl lithium and phenyl lithium; lithium alkylamides having 2 to 6 carbon atoms, such as lithium dimethylamide, lithium diethylamide and lithium diisopropylamide; metal hydrides such as lithium hydride and sodium hydride; metal alkoxides having 1 to 6 carbon atoms, such as lithium ethoxide, lithium tert-butoxide, sodium methoxide, sodium ethoxide and potassium tert-butoxide; amides such as lithium amide, potassium amide and sodium amide; inorganic bases such as lithium hydroxide, potassium hydroxide, sodium hydroxide, sodium carbonate, potassium carbonate and sodium hydrogen carbonate; and tertiary amines such as triethylamine, tri(n-propyl)amine, tri(n-butyl)amine, diisopropylethylamine, cyclohexyldimethylamine, pyridine, lutidine, xcex3-collidine, N,N-dimethylaniline, N-methylpiperidine, N-methylpyrrolidine and N-methylmorpholine. The reaction is carried out using 1 to 5 mol, preferably 1 to 3 mol, of compound (IV) per mol of compound (III). Reaction temperature is normally about xe2x88x9280 to 100xc2x0 C., preferably xe2x88x9250 to 60xc2x0 C. Reaction time is normally 1 minute to 72 hours, preferably 15 minutes to 24 hours, depending on the kinds of compounds (III) and (IV), the kind of solvent, reaction temperature etc. 
In the above formulas, R7 represents hydrogen or a lower alkyl group; R8 represents hydrogen or a hydroxyl group-protecting group; the other symbols have the same definitions as those shown above.
The lower alkyl group represented by R7 is exemplified by the same lower alkyl groups as those mentioned to exemplify the lower alkyl group used for R6 in method A.
The hydroxyl group-protecting group represented by R8 may be any one, as long as it does not interfere with the reaction; preferable examples thereof include ether-forming protecting groups such as methoxymethyl, benzyloxymethyl, tert-butoxymethyl, 2-methoxyethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methyithiomethyl, 2-tetrahydropyranyl, 4-methoxy-4-tetrahydropyranyl, 2-tetrahydrofranyl, benzyl, p-methoxybenzyl, p-nitrobenzyl, o-nitrobenzyl and trityl; silyl ether-forming protecting groups such as trimethylsilyl, triethylsilyl, triisopropylsilyl, isopropyldimethylsilyl, diethylisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, tribenzylsilyl, triphenylsilyl and methyldiphenylsilyl; and ester-forming protecting groups such as formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, pivaloyl and benzoyl.
When R8 in formula (V) is hydrogen, compound (V) or a salt thereof is reacted with compound (VI). The salt of compound (V) is exemplified by acid adduct salts with the acids mentioned to exemplify acids that form an acid adduct salt with compound (I). This reaction is normally carried out in a solvent and, if necessary, in the presence of a base. Such solvents and bases are identical to the solvents and bases mentioned for method A above. The reaction is carried out using 1 to 10 mol, preferably 1 to 5 mol, of compound (VI) per mol of compound (V) or salt thereof. Reaction temperature is normally about xe2x88x9230 to 200xc2x0 C., preferably xe2x88x9210 to 150xc2x0 C. Reaction time is normally 1 minute to 120 hours, preferably 15 minutes to 48 hours, depending on the kinds of compounds (V) and (VI), the kinds of solvent and base, reaction temperature etc.
Compound (I) can also be produced by producing compound (VIII) from compounds (V) and (VII) and cyclizing compound (VIII). This method involves the acylation of compound (VII) or a salt thereof with compound (V), a salt thereof or a reactive derivative thereof.
Specifically, free acid (V), a salt thereof (inorganic salt, organic salt) or a reactive derivative thereof (e.g., acid halide, acid azide, acid anhydride, mixed acid anhydride, active amide, active ester, active thioester etc.) is subjected to acylation reaction. Inorganic salts include alkali metal salts (e.g., sodium salt, potassium salt) and alkaline earth metal salts (e.g., calcium salt). Organic salts include, for example, trimethylamine salt, triethylamine salt, tert-butyldimethylamine salt, dibenzylmethylamine salt, benzyldimethylamine salt, N,N-dimethylaniline salt, pyridine salt and quinoline salt. Acid halides include, for example, acid chloride and acid bromide. Mixed acid anhydrides include mono-C1-4 alkylcarbonic acid mixed acid anhydrides (e.g., mixed acid anhydrides of free acid (V) and monomethylcarbonic acid, monoethylcarbonic acid, monoisopropylcarbonic acid, monoisobutylcarbonic acid, mono-tert-butylcarbonic acid, monobenzylcarbonic acid, mono(p-nitrobenzyl)carbonic acid, monoallylcarbonic acid etc.), C1-6 aliphatic carboxylic acid mixed acid anhydrides (e.g., mixed acid anhydrides of free acid (V) and acetic acid, cyanoacetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, pivalic acid, trifluoroacetic acid, trichloroacetic acid, acetoacetic acid etc.), C7-11 aromatic carboxylic acid mixed acid anhydrides (e.g., mixed acid anhydrides of free acid (V) and benzoic acid, p-toluic acid, p-chlorobenzoic acid etc.) and organic sulfonic acid mixed acid anhydrides (e.g., mixed acid anhydrides with methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid etc.). Active amides include amides with nitrogen-containing heterocyclic compounds [e.g., acid amides of free acid (V) and pyrazole, imidazole, benzotriazole etc.; these nitrogen-containing heterocyclic compounds may be substituted for by C1-4 alkyls (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl), C1-6 alkoxys (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy), halogen atoms (e.g., fluorine, chlorine, bromine), oxo, thioxo, C1-6 alkylthios (e.g., methylthio, ethylthio, propylthio, butylthio) etc.].
Active esters include, for example, organic phosphoric acid esters (e.g., diethoxyphosphoric acid esters, diphenoxyphosphoric acid esters), p-nitrophenyl ester, 2,4-dinitrophenyl ester, cyanomethyl ester, pentachlorophenyl ester, N-hydroxysuccinimide ester, N-hydroxyphthalimide ester, 1-hydroxybenzotriazole ester, 6-chloro-1-hydroxybenzotriazole ester and 1-hydroxy-1H-2-pyridone ester. Active thioesters include esters with aromatic heterocyclic thiol compounds [their heterocyclic rings may be substituted for by C1-4 alkyls (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl), C1-6 alkoxys (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy), halogen atoms (e.g., fluorine, chlorine, bromine), C1-6 alkylthios (e.g., methylthio, ethylthio, propylthio, butylthio) etc.] [e.g., 2-pyridylthiol ester, 2-benzothiazolylthiol ester].
The salt of compound (VII) is exemplified by salts with alkali metals (e.g., potassium, sodium, lithium), salts with alkaline earth metals (e.g., calcium, magnesium) and acid addition salts (acid adduct salts with the acids mentioned to exemplify acids that form an acid addition salt with compound (I)).
This reaction is normally carried in a solvent; a solvent that does not interfere with the reaction is chosen as appropriate. Such solvents include, for example, ethers such as dioxane, tetrahydrofuran, diethyl ether, tert-butyl methyl ether, diisopropyl ether and ethylene glycol-dimethyl ether; esters such as ethyl formate, ethyl acetate and butyl acetate; halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, trichlene and 1,2-dichloroethane; hydrocarbons such as n-hexane, benzene and toluene; amides such as formamide, N,N-dimethylformamide and N,N-dimethylacetamide; ketones such as acetone, methyl ethyl ketone and methyl isobutyl ketone; nitriles such as acetonitrile and propionitrile; dimethyl sulfoxide, sulfolane, hexamethylphosphoramide and water; these solvents are used as simple or mixed solvents. The amount of compound (VII) used is normally 1 to 10 mol, preferably 1 to 5 mol, per mol of compound (V). The reaction is normally carried out in the temperature range from xe2x88x9280 to 200xc2x0 C., preferably from xe2x88x9240 to 150xc2x0 C., and most preferably from xe2x88x9230 to 100xc2x0 C. Reaction time is normally 1 minute to 72 hours, preferably 15 minutes to 24 hours, depending on the kinds of compounds (V) and (VII), the kind of solvent (also mixing ratio in the case of a mixed solvent), reaction temperature etc. When compound (V) is used as an acid halide, the reaction can be carried out in the presence of a deoxidizer to remove the released hydrogen halide from the reaction system. Such deoxidizers include, for example, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate and sodium hydrogen carbonate; tertiary amines such as triethylamine, tripropylamine, tributylamine, cyclohexyldimethylamine, pyridine, lutidine, xcex3-collidine, N,N-dimethylaniline, N-methylpiperidine, N-methylpyrrolidine and N-methylmorpholine; and alkylene oxides such as propylene oxide and epichlorohydrin.
Compound (VIII) can be then cyclized to yield compound (I) after the hydroxyl group-protecting group R8 is removed as necessary. Depending on the kind of protecting group, this deprotection reaction can be carried out by a known method chosen as appropriate. For example, deprotection can be achieved with an acid (e.g., formic acid, acetic acid, propionic acid, hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid) or by catalytic reduction [Raney nickel, platinum, palladium, rhodium, or the like, for example, used as a catalyst at normal pressure or increased pressure (2 to 100 atm)] in the case of ether-forming protecting groups, with one of the above-mentioned acids or a Lewis acid (e.g., zinc chloride, zinc bromide, aluminum chloride, titanium chloride) or a fluoride (e.g., potassium fluoride, sodium fluoride, tetraethylammonium fluoride, tetra-n-butylammonium fluoride) in the case of silyl ether-forming protecting groups, or with a base (e.g., potassium hydrogen carbonate, sodium hydrogen carbonate, potassium carbonate, sodium carbonate, lithium hydroxide, potassium hydroxide, sodium hydroxide) in the case of ester-forming protecting groups. The reaction is normally carried out in a solvent; such solvents are exemplified by the solvents used for method A.
In the case of ether-forming protecting groups or silyl ether-forming protecting groups, the amount of acid or Lewis acid used is normally 0.001 to 100 mol, preferably 0.01 to 50 mol. per mol of compound (V). Reaction temperature is normally xe2x88x9250 to 150xc2x0 C., preferably xe2x88x9220 to 100xc2x0 C. Reaction time is normally 1 minute to 72 hours, preferably 15 minutes to 48 hours.
In the case of ester-forming protecting groups, the amount of base used is normally 0.01 to 50 mol, preferably 0.1 to 20 mol, per mol of compound (V). Reaction temperature is normally xe2x88x9220 to 150xc2x0 C., preferably xe2x88x9210 to 100xc2x0 C. Reaction time is normally 1 minute to 72 hours, preferably 15 minutes to 48 hours.
Compound (VIII) thus deprotected is cyclized to compound (I). This reaction is normally carried out in a solvent. Such solvents are exemplified by the solvents used for method A. Reaction temperature is normally xe2x88x9210 to 200xc2x0 C., preferably xe2x88x925 to 150xc2x0 C. In this reaction, a base may be used as a catalyst; such bases are exemplified by the bases used for method A. To promote the reaction, there may be used, for example, 2-chloro-3-methylbenzoxazolium tetrafluoroborate, 2-chloro-3-ethylbenzoxazolium tetrafluoroborate, 2-chloro-3-methylbenzothiazolium tetrafluoroborate, 2-chloro-3-ethylbenzothiazolium fluoroborate, 2-chloro-1-methylpyridinium tetrafluoroborate and 2-chloro-1-ethylpyridinium tetrafluoroborate. The amount of reaction promoter used is normally 1 to 10 mol, preferably 1 to 3 mol, per mol of compound (VIII). A base is also used when a reaction promoter is used. Such bases are exemplified by the bases used for method A. Reaction temperature is normally xe2x88x9230 to 150xc2x0 C., preferably xe2x88x9220 to 100xc2x0 C. Reaction time is normally 1 minute to 72 hours, preferably 15 minutes to 48 hours.
When one of R3 and R4 is an acyl group, an esterified carboxyl group or a carbamoyl group that may have a substituent, compound (I) can be produced by methods C, D and E below.
Of the compounds of formula (I), compound (Ib), which has an acyl group for R3 or R4, can be produced by method C. 
In these formulas, R4a represents an acyl group; R4b represents a group resulting from removal of the carbonyl group or sulfonyl group from an acyl group; the other symbols have the same definitions as those shown above.
The acyl group represented by R4a means an acyl group represented by R4; the xe2x80x9cacyl groupxe2x80x9d in the xe2x80x9cgroup resulting from removal of the carbonyl group or sulfonyl group from an acyl groupxe2x80x9d represented by R4b means an acyl group represented by R4.
In this reaction, compound (Ia) or a salt thereof can be acylated with compound (IX) or (X) or a reactive derivative thereof to yield compound (Ib). The salt of compound (Ia) is exemplified by the same acid adduct salts as those mentioned to exemplify the salt of compound (I). The reactive derivative of compound (IX) is exemplified by the reactive derivatives mentioned for method B. The reactive derivative of compound (X) is exemplified by sulfonic acid halides (e.g., sulfonyl bromide, sulfonyl chloride) and sulfonic anhydride; the reaction is carried out by the method described for method B or a modification thereof.
In the compounds of formula (I), compound (Ic), which has an esterified carboxyl group for R4, can be produced by method D. 
In these formulas, R4c represents an esterified carboxyl group; Q represents a halogen atom; the other symbols have the same definitions as those shown above.
R4c is any one of the carboxyl groups that may be esterified, represented by R4, except the free carboxyl group.
The halogen represented by Q is exemplified by fluorine, chlorine, bromine and iodine. This reaction is carried out by reacting compound (Ia) or a salt thereof and compound (XI). The salt of compound (Ia) is exemplified by the acid adduct salts mentioned to exemplify the acid adduct salt of compound (Ia) for reaction D above. This reaction is normally carried out in a solvent; such solvents are exemplified by the solvents used for method B. In this reaction, a hydrogen halide is released. To remove the hydrogen halide, the reaction can be carried out in the presence of an acid scavenger. Such acid scavenger includes, for example, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate and sodium hydrogen carbonate; tertiary amines such as triethylamine, tripropylamine, tributylamine, cyclohexyldimethylamine, pyridine, lutidine, xcex3-collidine, N,N-dimethylaniline, N-methylpiperidine, N-methylpyrrolidine and N-methylmorpholine; and alkylene oxides such as propylene oxide and epichlorohydrin.
The amount of compound (XI) used is normally 1 to 20 moll preferably 1 to 10 mol, per mol of compound (Ia). Reaction temperature is normally xe2x88x9230 to 120xc2x0 C., preferably xe2x88x9220 to 80xc2x0 C. Reaction time is normally 1 minute to 72 hours, preferably 15 minutes to 48 hours.
Of the compounds of formula (I), compound (Id), which has a carbamoyl group which may be substituted, can be produced by method E. 
In these formulas, R4d represents a carbamoyl group which may be substituted; R9, R10 and R11, whether identical or not, represent hydrogen or one of the substituents mentioned to exemplify the substituent for the carbamoyl group represented by R4, which may be substituted; the other symbols have the samel definitions as those shown above.
In this method, compound (Ic) or a salt thereof can be reacted with compound (XII) to yield compound (Id). The salt of compound (Ic) is exemplified by the same acid addition salts as those mentioned to exemplify the salt of compound (I). This reaction is normally carried out in a solvent; such solvents are exemplified by the solvents used for method A. The amount of compound (XII) used is normally 1 to 100 mol, preferably 1 to 30 mol, per mol of compound (Ic). Reaction temperature is normally xe2x88x9230 to 200xc2x0 C., preferably xe2x88x9210 to 100xc2x0 C. Reaction time is normally 1 minute to 72 hours, preferably 15 minutes to 48 hours.
Compound (Id) can also be produced by reacting compound (Ia) with isocyanate derivative (XIII). The reaction is normally carried out in a solvent. Said solvent may be any one, as long as it does not interfere with the reaction. For example, ethers such as dioxane, tetrahydrofuran, diethyl ether, tert-butyl ether, diisopropyl ether and ethylene glycol-dimethyl ether; esters such as ethyl formate, ethyl acetate and n-butyl acetate; halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, trichlene and 1,2-dichloroethane; hydrocarbons such as n-hexane, benzene and toluene; amides such as formamide, N,N-dimethylformamide and N,N-dimethylacetamide; ketones such as acetone, methyl ethyl ketone and methyl isobutyl ketone; nitriles such as acetonitrile and propionitrile; dimethyl sulfoxide, sulfolane, hexamethylphosphoramide are used as simple or mixed solvents.
The amount of compound (XIII) used is normally 1 to 30 mol, preferably 1 to 15 mol, per mol of compound (Ia). Reaction temperature is normally xe2x88x9220 to 150xc2x0 C., preferably xe2x88x9210 to 100xc2x0 C. Reaction time is normally 1 minute to 72 hours, preferably 15 minutes to 48 hours. 
In these formulas, R12 represents hydrogen, a lower alkyl group, a cycloalkyl group, an aralkyl group or an acyl group; R13 and R14, whether identical or not, represent hydrogen or a lower alkyl group; R15 represents hydrogen, a lower alkyl group or an aralkyl group; R16 represents a lower alkyl group or an aryl group; the other symbols have the same definitions as those shown above.
The lower alkyl group represented by R12 in formula (XIV) is exemplified by alkyl groups having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl and 2-ethylbutyl, with preference given to those having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl and isobutyl. The cycloalkyl group represented by R12 is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and cyclononyl, with preference given to cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The aralkyl group represented by R12 is exemplified by benzyl, phenethyl and phenylpropyl.
The acyl group represented by R12 is exemplified by aliphatic acyl groups such as alkanoyl groups, alkenoyl groups, cycloalkanecarbonyl groups and alkanesulfonyl groups; aromatic acyl groups such as aroyl groups, arylalkanoyl groups, arylalkenoyl groups and arenesulfonyl groups; heterocyclic aromatic acyl groups such as aromatic heterocyclic carbonyl groups and aromatic heterocyclic alkanoyl groups; and non-aromatic heterocyclic carbonyl groups (aliphatic heterocyclic carbonyl groups).
xe2x80x9cAlkanoyl groupsxe2x80x9d mean alkylcarbonyl groups, preferable examples thereof including lower alkanoyl groups having 1 to 8 carbon atoms, such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl and hexanoyl.
xe2x80x9cAlkenoyl groupsxe2x80x9d mean alkenylcarbonyl groups, preferable examples thereof including C3-6 alkenoyl groups such as acryloyl, methacryloyl, crotonoyl and isocrotonoyl.
xe2x80x9cCycloalkanecarbonyl groupsxe2x80x9d mean cycloalkylcarbonyl groups, preferable examples thereof including those having 4 to 7 carbon atoms, such as cyclopropanecarbonyl groups, cyclobutanecarbonyl groups, cyclopentanecarbonyl groups and cyclohexanecarbonyl groups.
xe2x80x9cAlkanesulfonyl groupsxe2x80x9d mean alkylsulfonyl groups, preferable examples thereof including those having 1 to 4 carbon atoms, such as mesyl, ethanesulfonyl and propanesulfonyl.
xe2x80x9cAroyl groupsxe2x80x9d mean arylcarbonyl groups, preferable examples thereof including those having 7 to 11 carbon atoms, such as benzoyl, p-toluoyl, 1-naphthoyl and 2-naphthoyl.
xe2x80x9cArylalkanoyl groupsxe2x80x9d mean alkylcarbonyl groups, substituted for by an aryl group, preferable examples thereof including C6-8 aryl-C2-5 alkanoyl groups such as phenylacetyl, phenylpropionyl hydroatropoyl and phenylbutyryl.
xe2x80x9cArylalkenoyl groupsxe2x80x9d mean alkenylcarbonyl groups substituted for by an aryl group, preferable examples thereof including C6-8 aryl-C3-5 alkenoyl groups such as cinnamoyl and atropoyl.
xe2x80x9cArenesulfonyl groupsxe2x80x9d mean arylsulfonyl groups, preferable examples thereof including those having 6 to 8 carbon atoms, such as benzenesulfonyl and p-toluenesulfonyl.
Preferable examples of xe2x80x9caromatic heterocyclic carbonyl groupsxe2x80x9d include furoyl, thenoyl, nicotinoyl, isonicotinoyl, pyrrolecarbonyl, oxazolecarbonyl, thiazolecarbonyl, imidazolecarbonyl and pyrazolecarbonyl.
xe2x80x9cAromatic heterocyclic alkanoyl groupsxe2x80x9d mean alkylcarbonyl groups substituted for by an aromatic heterocyclic group, preferable examples thereof including aromatic heterocyclic ring-C2-5 alkanoyl groups such as thienylacetyl, thienylpropanoyl, furylacetyl, thiazolylacetyl, 1,2,4-thiadiazolylacetyl and pyridylacetyl.
Preferable examples of xe2x80x9cnon-aromatic heterocyclic carbonyl groupsxe2x80x9d include aliphatic heterocyclic carbonyls such as azetidinylcarbonyl, pyrrolidinylcarbonyl and piperidinylcarbonyl.
The lower alkyl group represented by R13, R14, R15 or R16 in formulas (XIV), (XV) and (XVI) is exemplified by lower alkyl groups represented by R12. The aralkyl group represented by R15 is exemplified by aralkyl groups represented by R12. The aryl group represented by R16 is exemplified by phenyl, naphthyl, anthryl, phenanthryl and acenaphthylenyl groups, with preference given to phenyl and naphthyl. These aryl groups may have 1 to 5 substituents. Such substituents include alkyl groups having 1 to 3 carbon atoms (e.g., methyl, ethyl, propyl), alkoxy groups having 1 to 3 carbon atoms (e.g., methoxy, ethoxy, propoxy) and halogen atoms (e.g., fluorine, chlorine, bromine, iodine).
In this method, compounds (XIV) and (XV) are reacted in the presence of compound (XVI) to yield compound (XVII).
This reaction is normally carried out in a solvent; a solvent that does not interfere with the reaction is chosen as appropriate. Such solvents include, for example, alcohols such as methanol, ethanol, propanol, isopropanol, butanol and tert-butanol; ethers such as dioxane, tetrahydrofuran, diethyl ether, tert-butyl methyl ether, diisopropyl ether and ethylene glycol-dimethyl ether; esters such as ethyl formate, ethyl acetate and n-butyl acetate; halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, trichlene and 1,2-dichloroethane; hydrocarbons such as n-hexane, benzene and toluene; amides such as formamide, N,N-dimethylformamide and N,N-dimethylacetamide; ketones such as acetone, methyl ethyl ketone and methyl isobutyl ketone; nitriles such as acetonitrile and propionitrile; dimethyl sulfoxide, sulfolane, hexamethylphosphoramide and water; these solvents are used as simple or mixed solvents.
Reaction temperature is normally xe2x88x9280 to 150xc2x0 C., preferably xe2x88x9250 to 120xc2x0 C. The amount of each of compounds (XV) and (XVI) used is normally 1 to 5 mol, preferably 1 to 3 mol, per mol of compound (XIV).
The ester of compound (XVII) is then subjected to hydrolysis, hydrogenolysis, or the like, to yield compound (XVIII).
This hydrogenolysis reaction is normally carried out in a solvent; a solvent that does not interfere with the reaction is chosen as appropriate. Such solvents include, for example, alcohols such as methanol, ethanol, propanol, isopropanol, butanol and tert-butanol; ethers such as dioxane, tetrahydrofuran, diethyl ether, tert-butyl methyl ether, diisopropyl ether and ethylene glycol-dimethyl ether; halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, trichlene and 1,2-dichloroethane; hydrocarbons such as n-hexane, benzene and toluene; amides such as formamide, N,N-dimethylformamide and N,N-dimethylacetamide; ketones such as acetone, methyl ethyl ketone and methyl isobutyl ketone; nitrites such as acetonitrile and propionitrile; dimethyl sulfoxide, sulfolane, hexamethylphosphoramide and water; these solvents are used as simple or mixed solvents.
This reaction is carried out in the presence of a base. Preferably used bases include metal hydroxides such as lithium hydroxide, potassium hydroxide, sodium hydroxide and barium hydroxide, and metal carbonates such as potassium carbonate, sodium carbonate and barium carbonate. The amount of base used is normally 1 to 30 mol, preferably 1 to 10 mol, per mol of compound (XVII). Reaction temperature is normally xe2x88x9230 to 150xc2x0 C., preferably xe2x88x9210 to 120xc2x0 C. Reaction time is normally 15 minutes to 48 hours, preferably 30 minutes to 24 hours.
When compound (XVIII) is produced by a hydrogenolysis reaction, the reaction is normally carried out using a catalyst. This catalyst is preferably one for catalytic reduction reaction, exemplified by platinum catalysts (e.g., platinum oxide, platinum black, platinum-carbon), palladium catalysts (e.g., palladium chloride, palladium-carbon, palladium-calcium carbonate, palladium-barium sulfate), rhodium catalysts (e.g., rhodium-carbon, rhodium-alumina) and ruthenium catalysts (e.g., ruthenium oxide, ruthenium-carbon), with greater preference given to palladium catalysts. The reaction is normally carried out in a solvent; a solvent that does not interfere with the reaction is chosen as appropriate. Such solvents include, for example, alcohols such as methanol, ethanol, propanol, isopropanol, butanol and tert-butanol; ethers such as dioxane, tetrahydrofuran, diethyl ether, tert-butyl methyl ether, diisopropyl ether and ethylene glycol-dimethyl ether; esters such as ethyl formate, ethyl acetate and n-butyl acetate; hydrocarbons such as n-hexane, benzene and toluene; amides such as formamide, N,N-dimethylformamide and N,N-dimethylacetamide; and water; these solvents are used as simple or mixed solvents.
Reaction temperature is normally xe2x88x9210 to 120xc2x0 C., preferably 0 to 100xc2x0 C. Although this reaction is normally carried out at normal pressure, it may be carried out at increased pressure in some cases. Such pressure is preferably 1 to 200 atm.
Compound (XVIII) can be decarbonized by heating to yield compound (I). This reaction is normally carried out in a solvent; a solvent that does not interfere with the reaction is chosen as appropriate. Such solvents include, for example, alcohols such as methanol, ethanol, propanol, isopropanol, butanol and tert-butanol; ethers such as dioxane, tetrahydrofuran, diethyl ether, tert-butyl methyl ether, diisopropyl ether and ethylene glycol-dimethyl ether; esters such as ethyl formate, ethyl acetate and n-butyl acetate; halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, trichlene and 1,2-dichloroethane; hydrocarbons such as n-hexane, benzene and toluene; amides such as formamide, N,N-dimethylformamide and N,N-dimethylacetamide; ketones such as acetone, methyl ethyl ketone and methyl isobutyl ketone; nitriles such as acetonitrile and propionitrile; dimethyl sulfoxide, sulfolane, hexamethylphosphoramide and water; these solvents are used as simple or mixed solvents.
Reaction temperature is normally 0 to 180xc2x0 C., preferably 10 to 150xc2x0 C. Reaction time is normally 5 minutes to 24 hours, preferably 10 minutes to 12 hours.
When a compound involved in each reaction described above has an amino group, a carboxyl group or a hydroxyl group as a substituent, the group may incorporate a protecting group in common use in peptide chemistry and other fields; the desired compound can be obtained by removing the protecting group as necessary after reaction.
Useful amino group-protecting groups include, for example, formyl group, C1-6 alkylcarbonyl groups (e.g., acetyl, ethylcarbonyl), benzyl group, tert-butyloxycarbonyl group, benzyloxycarbonyl group, 9-fluorenylmethyloxycarbonyl group, allyloxycarbonyl group, phenylcarbonyl group, C1-6 alkyloxycarbonyl groups (e.g., methoxycarbonyl, ethoxycarbonyl), C7-10 aralkylcarbonyl groups (e.g., benzylcarbonyl), trityl group, phthaloyl group and N,N-dimethylaminomethylene group. These groups may be substituted by 1 to 3 halogen atoms (e.g., fluorine, chlorine, bromine), nitro group etc.
Useful carboxyl group-protecting groups include C1-6 alkyl groups (e.g., methyl, ethyl, propyl, isopropyl, butyl, tert-butyl), phenyl group, silyl group, benzyl group and allyl group. These groups may be substituted for by 1 to 3 halogen atoms (e.g., fluorine, chlorine, bromine), nitro group etc.
Useful hydroxyl group-protecting groups include methoxymethyl group, allyl group, tert-butyl group, C7-10 aralkyl groups (e.g., benzyl), formyl group, C1-6 alkylcarbonyl groups (e.g., acetyl, ethylcarbonyl), benzoyl group, C7-10 aralkylcarbonyl groups (e.g., benzylcarbonyl), pyranyl group, furanyl group and trialkylsilyl groups. These groups may be substituted by 1 to 3 halogen atoms (e.g., fluorine, chlorine, bromine), C1-6 alkyl groups (e.g., methyl, ethyl, propyl, isopropyl, butyl, tert-butyl), phenyl group, C7-10 aralkyl groups (e.g., benzyl), nitro group etc.
These protecting groups can be removed by commonly known methods or modifications thereof, including those using acids, bases, reduction, ultraviolet rays, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate etc.
When a compound is obtained in a free form by each of the above-described reactions of the present invention, it may be converted to a salt by a conventional method; when it is obtained as a salt, it may be converted to a free form or another salt by a conventional method.
Compound (I) thus obtained can be isolated and purified from the reaction mixture by commonly known means such as extraction, concentration, neutralization, filtration, recrystallization, column chromatography and thin-layer chromatography.
A salt of compound (I) can be produced by, for example, adding one of the above-described inorganic acids or organic acids to compound (I) by a commonly known means.
Compounds (III) and (IV), used as starting compounds in method A above, can be produced by, for example, the method described in U.S. Pat. No. 4,584,385 or a method based thereon.
Compound (V), used as a starting compound in method B above, can be produced by, for example, the method described in the Journal of Medicinal Chemistry, 21, 82 (1978) or Chemistry Letters, 166 (1980) or a method based thereon; compound (VI) can be produced by, for example, the method described in the Journal of Organic Chemistry, 42, 3608 (1977) or a method based thereon; compound (VII) can be produced by the method described in the Journal of the Chemical Society, 95, 132 (1909) or a method based thereon.
In addition to the above-mentioned processes, compound (I) can also be produced by the method described in U.S. Pat. No. 4,584,385 or a method based thereon.
Although compound (I) can be produced by chemical processes as described above, it can also be produced using microorganisms. Of the compounds of formula (I), indolmycin, can be produced by, for example, the methods described in the literature [K.V. Rao, Antibiotics and Chemotherapy (Washington, D.C.), 10, 312 (1960); W.S. Marsh et al., ibid., 10, 316 (1960); Schach von Wittenau, M. et al., J. Am. Chem. Soc. 83, 4678 (1961), ibid., 85, 3425 (1963)], using as producer strains Streptomyces griseus subsp. griseus ATCC 1264 (American Type Culture Collection Catalogue of Bacteria and Bacteriophages, 18th edition, 1992) etc. Streptomyces sp. HC-21, a new strain, can also be used as a producer strain.
The microorganism used for the method of indolmycin production of the present invention is the Streptomyces sp. HC-21 strain (hereinafter also referred to as xe2x80x9cHC-21 strainxe2x80x9d) isolated from a soil sample from Tenninkyo, Asahikawa-shi, Hokkaido, Japan.
According to the method described in the International Journal of Systematic Bacteriology, 16(3), 313-340 (1960), the EC-21 strain is characterized as follows: All findings on medium were obtained during 14 days of cultivation and observation at 28xc2x0 C., unless otherwise stated.
(I) Morphological Characteristics
The aerial mycelia elongate in simple branches from well elongated and branched substrate mycelia, with gently waved or key-shaped spore chains (normally 10 to 50 spores or more) on their tips. No whirls are noted. Spores are cylindrical (1.1 to 1.2xc3x971.4 to 1.5 xcexcm) and have a spiny surface.
(II) Nature in Culture
Degree of growth (G), growth and color tone of aerial mycelia (AM), back face color tone (R), presence or absence and color tone of soluble pigment (SP) etc. on various media are described below. For the description of color, standard color tone symbols in parentheses are based on the Color Harmony Manual of Container Corporation of America, 4th edition, 1958.
(IV) Cell Analysis
Analysis in accordance with the method of Hasegawa et al. [Journal of General Applied Microbiology 29, 319-322 (1983)] identified the diaminopimelic acid in the hydrochloric acid hydrolyzate of cells as the LL-configuration.
Judging from the results shown above, specifically the light yellowish brown to grayish brown aerial mycelia, gently waved or key-shaped spore chains, spiny spore surfaces, diaminopimelic acid in the LL-configuration, and other findings, it is evident that this strain belongs to the genus Streptomyces; the strain was designated Streptomyces sp. HC-21.
The Streptomyces sp. HC-21 strain as such is characterized by the capability of L-rhamnose assimilation and spiny spore surfaces.
The Streptomyces sp. HC-21 strain as such has been deposited under accession number IFO-15984 at the Institute for Fermentation, Osaka (foundation), since June 12, 1996, and under accession number FERM BP-5571 at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry of the Japan (1-3, Higashi 1-chome, Yatabe, Tsukuba City, Ibaraki Prefecture), since Jun. 25, 1996.
The bacteria of the genus Streptomyces can undergo variation, naturally or by mutagens, as a general nature of microorganisms. Even the various variants obtained by, for example, irradiation with radiations such as X rays, gamma rays and ultraviolet rays, single spore separation, treatment with various chemicals, cultivation on drug-containing media, and other means, or naturally-occurring mutants, are all usable for the method of the present invention, as long as they are capable of producing indolmycin.
Although the culture medium for the method of the present invention may be liquid or solid, as long as it contains nutrient sources usable by the strain used, a liquid medium is preferred for large-scale treatment. The medium is supplemented as appropriate with assimilable nutrient sources, digestible nitrogen sources, inorganic substances, and trace nutrients.
Carbon sources include, for example, glucose, lactose, sucrose, maltose, dextrin, starch, glycerol, mannitol, sorbitol, oils and fats (e.g., soybean oil, olive oil, rice bran oil, sesame oil, lard oil, chicken oil); nitrogen sources include, for example, meat extract, yeast extract, dry yeasts, soybean flour, corn steep liquor, peptone, cottonseed flour, blackstrap molasses, urea, ammonium salts (e.g., ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate) and others. Also used as appropriate are salts containing sodium, potassium, calcium, magnesium etc., metal salts such as those of iron, manganese, zinc, cobalt, nickel etc., salts of phosphoric acid, boric acid etc., and salts of organic acids such as acetic acid and propionic acid. Additionaliy, amino acids (e.g., glutamic acid, aspartic acid, alanine, lysine, valine, methionine, proline), vitamins (e.g., B1, B2, nicotinic acid, B12, C), nucleic acids (e.g. purine, pyrimidine and derivatives thereof) etc. may be contained. It is of course common practice to add inorganic or organic acids, alkalis, buffers etc. for regulation of the medium""s pH, and appropriate amounts of oils and fats, surfactants etc. for defoaming.
Cultivation may be achieved by standing culture, shaking culture, spinner culture, or the like. For large-scale treatment, submerged spinner culture is of course desirable.
Although culturing conditions vary depending on the condition and composition of the medium, the kind of strain, and the means of cultivation, it is normally recommended that temperature and initial pH be 15 to 26xc2x0 C. and about 5 to 9, respectively. It is desirable that temperature in the middle stage of cultivation and initial pH be 20 to 25xc2x0 C. and about 6 to 8, respectively. Duration of cultivation also varies depending on the above-mentioned conditions but it is recommended that cultivation be continued until the concentration of the desired bioactive substance reaches maximum. It normally takes about 1 to 10 days in the case of shaking culture or spinner culture using a liquid medium.
The resulting bioactive substance indolmycin can be extracted and purified from the culture on the basis of its chemical nature.
Because indolmycin is produced in the culture broth and cells, it can be purified by separating the culture broth and cells by filtration or centrifugation from the culture, extracting it from the resulting filtrate or centrifugal supernatant using an organic solvent, or extracting it from cells using an organic solvent, and isolating it from each extract or the combined extract.
For industrial purposes, it is advantageous to purify indolmycin from the extract obtained by adding an organic solvent such as methanol, acetone, butanol or ethyl acetate directly to the culture, with the cell separation operation omitted.
Because indolmycin is a weakly basic oil-soluble substance, its collection from the culture broth permits the use of means of separation and purification in common use for collection of related microbial metabolites. For example, methods based on solubility differences from impurity substances and chromatographies using various carriers such as activated charcoal nonionic high porous resin, silica gel, alumina and dextran gel can be used singly or in combination.
The method of isolating and collecting indolmycin from the culture is hereinafter described specifically. First, cells are removed by filtration from the culture broth; the resulting supernatant is adjusted to appropriate pH; a solvent such as ethyl acetate is added, followed by vigorous stirring, to yield an ethyl acetate layer. The organic layer obtained is sequentially washed with alkali, acid and water, after which it is concentrated; the resulting concentrate is subjected to silica gel column chromatography. Useful developing solvents include, for example, chloroform-methanol or hexane-acetone mixed solvents. After the effective fractions are combined and concentrated, the concentrate is subjected to Sephadex LH-20 chromatography. Useful developing solvents are methanol and mixed solvents such as hexane-toluene-methanol and hexane-methylene chloride-methanol. After concentration, the eluate containing the effective fractions is purified by preparative high performance liquid chromatography. The column packing used here is ODS-SH343 S-15 (produced by Yamamura Kagaku Kenkyujo); the solvent system used is a combination of 0.02 M phosphate buffer (pH 6.3) and 26% CH3CN.